CN110106261A - The combination of the SNP marker of Jiaxing Black Pig and raw meat product and identification method - Google Patents
The combination of the SNP marker of Jiaxing Black Pig and raw meat product and identification method Download PDFInfo
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Abstract
The invention discloses a kind of combination of the SNP marker of Jiaxing Black Pig and raw meat product and identification methods, by the genomic DNA for extracting raw pork or meat products, it is sequenced through the laggard row agarose gel electrophoresis of PCR amplification and Sanger, according to the SNP genotype identification Jiaxing Black Pig and its meat products in sequencing result feature site；The Sanger sequencing, identifies site are as follows: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200 and pig13-111071937 specific mutagenesis occur at site；The present invention solves the problems, such as that there has been no Jiaxing Black Pig and its relevant identification methods of meat products in the prior art.
Description
Technical field
The present invention relates to a kind of food safety monitoring fields, and in particular to a kind of Jiaxing Black Pig and raw meat product
SNP marker combination and identification method.
Background technique
Taihu Lake basin local varieties pig mainly has: Erhualian, plum mountain pig, Fengjing pig, husky rhizome of Chinese monkshood pig, rice pig, Jiaxing Black
Pig, wherein plum mountain pig because body size is divided into middle plum mountain pig and little MeiShan pig.Taihu Lake basin local varieties pig is and each with root common source
There is the case where obscuring in production in tool feature, but because its appearance traits is close, especially piglet, and by traditional figure outside
It is larger that each kind of Taihu Lake basin local varieties pig is distinguished difficulty by looks cultivar identification method, is unfavorable for effectively carrying out guarantor
Kind and development and utilization work.On the other hand, Taihu Lake basin local varieties Meat is delicious and extensively get consumer reception, economic valence
Value is higher than the filial generations porks such as the common Duroc in market, long white, Large White, thus occurs above-mentioned filial generation in the market
The case where Taihu Lake basin local varieties pork sale of pork personation, and above-mentioned hybridization pork is adulterated in Taihu Lake basin place
The sales behavior of kind pork product (such as ham, meat stuffing etc.), but conventional method can not cut meat to slaughter and treated
Meat products carries out accurate ownership judgement.Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) is main
Refer to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level, there is site to enrich, be distributed extensively
It is general, genetic stability is high, it is representative, detection it is convenient and quick the features such as.And wherein, in certain kind (group) range
Interior, the SNP only occurred in a group is referred to as the special SNP of kind.The special SNP site screening of kind and Sites Combination method
Research is the key that carry out kind and product identification.
Third generation molecular marker SNP has variation abundant, low, steady to DNA sample requirement relative to preceding two generations molecular labeling
The advantages that qualitative high, measurement is accurate, detection method is easy and flux is high.Currently, third generation molecular marker SNP is widely used to
The fields such as paternity test, plant and animal species (strain) identification, genetic breeding.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to propose a kind of identification Jiaxing Black Pig and raw meat product
SNP marker combination and identification method, are identified using third generation molecular labeling and Sanger sequencing technologies identify Jiaxing Black Pig and life
Meat products solves the problems, such as to provide one kind there has been no Jiaxing Black Pig and its relevant identification method of meat products in the prior art
As a result the method for accurate, easy to operate, cheap identification Jiaxing Black Pig and its meat products and related primer special.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of combination of the SNP marker of Jiaxing Black Pig and raw meat product, comprising: pig2-60239356, pig8-5778954,
Pig7-37079960, pig13-16955200 and pig13-111071937；The pig2-60239356 indicates pig No. 2 dyeing
The 60239356th site of body, pig8-5778954 indicate the 5778954th site of No. 8 chromosomes of pig, pig7-37079960 table
Showing the 37079960th site of No. 7 chromosomes of pig, pig13-16955200 indicates the 16955200th site of No. 13 chromosomes of pig,
Pig13-111071937 indicates the 111071937th site of No. 13 chromosomes of pig.
Further, Jiaxing Black Pig and its raw meat product identification method based on SNP marker combination, are extracted to be identified first
Raw pork or meat products genomic DNA, be then sequenced, obtain through the laggard row agarose gel electrophoresis of PCR amplification and Sanger
There is specific mutagenesis when SNP marker combines at any three sites in the information that each detection site is combined to SNP marker
It is accredited as Jiaxing Black Pig and its meat products.
Further, the PCR amplification, the sequence such as SEQ ID NO.1 of the upstream and downstream primer at pig2-60239356
Shown in~2, the sequence of the upstream and downstream primer at pig8-5778954 is as shown in NO.3~4 SEQ ID, at pig7-37079960
Upstream and downstream primer sequence as shown in NO.5~6 SEQ ID, the sequence of the upstream and downstream primer at pig13-16955200 is such as
Shown in NO.7~8 SEQ ID, the sequence of the upstream and downstream primer at pig13-111071937 is as shown in NO.9~10 SEQ ID.
Further, sequencing product identification site comparative information is as shown in the table:
| SNP | REF | ALT |
| pig2-60239356 | C | T |
| pig8-5778954 | G | A |
| pig7-37079960 | G | A |
| pig13-16955200 | T | C |
| pig13-111071937 | G | T |
REF, which is represented, in table refers to genotype, and ALT represents mutated-genotype.
Further, the identification site information are as follows: the mutated-genotype of each detection site with refer to genotype, when
Pig detection site to be measured thinks that the site does not have Identification Significance when occurring with reference to genotype, when pig detection site to be measured is mutated
When genotype, it is believed that the site has Identification Significance.
The beneficial effects of the present invention are: compared with prior art, the present invention is mirror with the peculiar SNP site of Jiaxing Black Pig kind
Determine foundation, the method for identifying Jiaxing Black Pig is studied from molecular level, and with Sanger sequencing for main method for identifying molecules, it can
Jiaxing Black Pig and other pig variety (strain) mutual authentications are distinguished, and Jiaxing Black Pig and common Modern China can be identified area
Point, such as: little MeiShan pig, Fengjing pig, middle plum mountain pig, Erhualian, rice pig, husky rhizome of Chinese monkshood pig, Landrace, Large White, Duroc,
Pietrain, Berkshire etc..
Specific embodiment
The specific embodiment of the invention is described in further detail below.
The present invention relates to the SNP markers of a kind of Jiaxing Black Pig and raw meat product, comprising: pig2-60239356, pig8-
5778954, pig7-37079960, pig13-16955200 and pig13-111071937；The pig2-60239356 indicates pig
The 60239356th site of No. 2 chromosome, pig8-5778954 indicate the 5778954th site of No. 8 chromosomes of pig, pig7-
37079960 indicate the 37079960th sites of No. 7 chromosomes of pig, and pig13-16955200 indicates No. 13 chromosomes of pig the
16955200 sites, pig13-111071937 indicate the 111071937th site of No. 13 chromosomes of pig.
The present invention provides a kind of Jiaxing Black Pigs and raw meat product identification method based on the combination of above-mentioned SNP marker, first
Extract the genomic DNA of raw pork or meat products to be identified, then through the laggard row agarose gel electrophoresis of PCR amplification and
Sanger sequencing, obtains the information that SNP marker combines each detection site, occurs when SNP marker combines at any three sites
Specific mutagenesis can be accredited as Jiaxing Black Pig and its meat products.
The PCR amplification, the primer being directed to are as shown in table 1:
Table 1 expands site primer and resulting information
F represents upstream primer in table, and R represents downstream primer, and length represents standardized products length, F'-position generation
Table SNP site is in the position of amplified production.
The PCR amplification, reaction system are template DNA 1ng/uL, primer 1uL, H2O 3.8uL、2×Taq PCR
Masrer Mix 50uL；And/or the response procedures of PCR reaction are 95 DEG C of initial denaturation 2min, 95 DEG C of initial denaturation 30s, 60
DEG C annealing temperature 30s, 72 DEG C of extension 1min, cycle-index 30 times, 72 DEG C re-extend 10min.
The mass concentration of the Ago-Gel is 2%.
The gel electrophoresis, band length requirement is as shown in table 1.
It is as shown in table 2 that product identification site comparative information is sequenced:
Table 2 is sequenced product and identifies site comparative information
| SNP | REF | ALT |
| pig2-60239356 | C | T |
| pig8-5778954 | G | A |
| pig7-37079960 | G | A |
| pig13-16955200 | T | C |
| pig8-5778954 | G | T |
REF, which is represented, in table refers to genotype, and ALT represents mutated-genotype.
As shown in table 2, the mutated-genotype of each detection site with refer to genotype, when pig detection site to be measured is joined
Think that the site does not have Identification Significance when examining genotype, when mutated-genotype occurs in pig detection site to be measured, it is believed that the site
With Identification Significance, such as a pig A to be measured is in the site pig2-60239356, if sequencing data is C, then it is assumed that pig A to be measured
Do not have Identification Significance in the site pig2-60239356；If sequencing data is T, then it is assumed that pig A to be measured is in pig2-
60239356 sites have Identification Significance.
Since there are false positive misjudgement as appraisal basis for single locus, and it is higher to misjudge probability, so present invention benefit
Use Sites Combination as identification of M arker.
Each cultivar identification Sites Combination identification of M arker information is as shown in table 3.
3 identification marking combined information of table
As shown in table 3, Marker1-10 is the combination of Jiaxing Black Pig identification marking, such as when pig A to be measured has 10
When any one site mutation in Marker combination combines, then it is assumed that pig A to be measured is Jiaxing Black Pig.
The Jiaxing Black Pig meat products refers to that Jiaxing Black Pig is cut meat and salted down using Jiaxing Black Pig as prepared by Raw material processing
Product and cooked product.
5 parts of pig kind ear tissue sample to be measured are randomly selected, tissue DNA is extracted using SDS method.
PCR reaction amplification is carried out using primer pair DNA sample.
The reaction system of PCR reaction is 10uL system: template DNA 1ng/uL, primer 1uL, H2O 3.8uL、2×Taq
PCR Masrer Mix 50uL；And/or the response procedures of PCR reaction are 95 DEG C of initial denaturation 2min, 95 DEG C of initial denaturation 30s, 60
DEG C annealing temperature 30s, 72 DEG C of extension 1min, cycle-index 30 times, 72 DEG C re-extend 10min.
With 2% Ago-Gel, 1 × TAE buffer is that dielectrophoresis detects amplification, and Gel electrophoresis conditions are electricity
Flow 10A, 100 volts of voltage, the time 40 minutes.The amplified production of different primers is compared with the standardized products length in table 1, produces
Object length is in error range and consistent with standardized products length, then it is assumed that amplification is qualified.
Qualified sample pcr amplification product is subjected to Sanger sequencing, obtains the information of each detection site, sequencing is tied
Fruit is analyzed:
4 sample sequencing result SNP polymorphism analysis table of table
| Sample/SNP | REF | ALT | 1 | 2 | 3 | 4 | 5 |
| pig2-60239356 | C | T | √ | √ | |||
| pig8-5778954 | G | A | √ | √ | |||
| pig7-37079960 | G | A | √ | √ | |||
| pig13-16955200 | T | C | √ | √ | √ | √ | |
| pig13-111071937 | G | T | √ | √ |
REF, which is represented, in table refers to genotype, and ALT represents mutated-genotype, and √ representative detects mutated-genotype.
As shown in table 4, it can be obtained by sequencing data, if one pig to be measured is simultaneously only with single site as appraisal basis
Belong to multiple kinds.
Identified in the way of Sites Combination Marker: No. 1 pig pig2-60239356, pig8-5778954,
Pig7-37079960, pig13-16955200, pig13-111071937 site primer to mutation, meet Marker1,
The mirror of Marker2, Marker3, Marker4, Marker5, Marker6, Marker7, Marker8, Marker9, Marker10
Determine information, identifies that No. 1 pig is Jiaxing Black Pig；Mutation is being not detected in No. 2 pigs, does not meet Marker information, identifies that No. 2 pigs are not
Jiaxing Black Pig；No. 3 pigs are in the site pig8-5778954, pig7-37079960, pig13-16955200, pig13-111071937
It detects mutation, meets Marker4, Marker7, Marker9, Marker10, identify that No. 3 pigs are Jiaxing Black Pig；No. 4 pigs exist
Pig2-60239356, pig13-16955200 site primer do not meet Marker information, identify No. 4 Jiaxing Zhu Bushi to mutation
Black pig；No. 5 pigs, to mutation, do not meet Marker information, identify No. 5 Jiaxing Zhu Bushi in pig13-16955200 site primer
Pig.
The related patents that do not identified both at home and abroad about Jiaxing Black Pig kind and its meat products kind (strain) at present, by third
Apply to Jiaxing Black Pig and its meat products kind (strain) identification for molecular labeling, has filled up the vacancy in market, efficiently solved
The false distinguishing problem of Jiaxing Black Pig (strain).The identification method utilizes first generation molecular labeling (RFLP) and the second generation relative to existing
Molecular labeling (SSR) does the patent of pig kind identification, has the advantages that operate simpler, result more accurately, rapidly and efficiently.Together
When the present invention overcome preceding two generations molecular labeling using third generation molecular marker SNP and can be used the few disadvantage in site, relatively preceding two
For the authentication technique of molecular labeling, discrimination method is also simplified.
Above-mentioned specific implementation can by those skilled in the art under the premise of without departing substantially from the principle of the invention and objective with difference
Mode carry out local directed complete set to it, protection scope of the present invention is subject to claims and not by above-mentioned specific implementation institute
Limit, each implementation within its scope is by the constraint of the present invention.
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Claims (5)
1. the SNP marker of a kind of Jiaxing Black Pig and raw meat product combines characterized by comprising pig2-60239356, pig8-
5778954, pig7-37079960, pig13-16955200 and pig13-111071937；The pig2-60239356 indicates pig
The 60239356th site of No. 2 chromosome, pig8-5778954 indicate the 5778954th site of No. 8 chromosomes of pig, pig7-
37079960 indicate the 37079960th sites of No. 7 chromosomes of pig, and pig13-16955200 indicates No. 13 chromosomes of pig the
16955200 sites, pig13-111071937 indicate the 111071937th site of No. 13 chromosomes of pig.
2. a kind of Jiaxing Black Pig and its raw meat product identification method, feature based on the combination of SNP marker described in claim 1 exists
In, extract the genomic DNA of raw pork or meat products to be identified first, then after PCR amplification carry out Ago-Gel electricity
Swimming and Sanger sequencing, obtain the information that SNP marker combines each detection site, when SNP marker combines at any three sites
There is specific mutagenesis, Jiaxing Black Pig and its meat products can be accredited as.
3. according to the method described in claim 2, it is characterized in that, the PCR amplification, at pig2-60239356 up and down
The sequence of primer is swum as shown in NO.1~2 SEQ ID, the sequence of the upstream and downstream primer at pig8-5778954 such as SEQ ID
Shown in NO.3~4, the sequence of the upstream and downstream primer at pig7-37079960 is as shown in NO.5~6 SEQ ID, pig13-
The sequence of upstream and downstream primer at 16955200 as shown in NO.7~8 SEQ ID, draw by the upstream and downstream at pig13-111071937
The sequence of object is as shown in NO.9~10 SEQ ID.
4. according to the method described in claim 2, it is characterized in that, sequencing product identification site comparative information is as shown in the table:
REF, which is represented, in table refers to genotype, and ALT represents mutated-genotype.
5. according to the method described in claim 4, it is characterized in that, the identification site information are as follows: each detection site
Mutated-genotype thinks that meaning is not identified in the site when pig detection site to be measured occurs with reference to genotype with reference to genotype
Justice, when mutated-genotype occurs in pig detection site to be measured, it is believed that the site has Identification Significance.
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| CN201910462678.4A CN110106261B (en) | 2019-05-30 | 2019-05-30 | SNP marker combination and identification method of Jiaxing black pig and raw meat product |
| PCT/CN2019/130974 WO2020238218A1 (en) | 2019-05-30 | 2019-12-31 | Snp marker combination and identification method for jiaxing black pig and raw meat product |
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| WO2020238218A1 (en) | 2020-12-03 |
| CN110106261B (en) | 2020-11-24 |
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